Separate urinary bladder and external urethral sphincter neurons in the central nervous system of the rat: simultaneous labeling with two immunohistochemically distinguishable pseudorabies viruses

Abstract

This work examines the distribution, in the central nervous system, of virus-labeled neurons from the rat urinary bladder and the external urethral sphincter simultaneously within the same tissue sections. Two immunohistochemically distinct pseudorabies virus strains were injected into male Sprague–Dawley rats (∼280 g). One virus was injected into the bladder and the other into the external urethral sphincter. After incubation intervals of 2, 2.5 and 3 days, sections from the spinal cord and brain were treated immunohistochemically to detect cells which were labeled separately by each virus or were labeled by both viruses. The major result of these experiments is that each strain of virus labeled a separate population of neurons and that some neurons were labeled by both strains. In the lumbosacral cord, 3 days post-infection, neurons labeled by virus from the external urethral sphincter were found in Onuf’s nucleus, the dorsal gray commissure, and the superficial dorsal horn. Neurons labeled by virus from the urinary bladder were found in the L6–S1 and L1–L2 spinal cord segments within the dorsal gray commissure, the intermediolateral area and the superficial dorsal horn. Double-labeled interneurons were mainly located in the dorsal gray commissure although some were also found in the intermediolateral area and the superficial dorsal horn. In the medulla, external urethral sphincter neurons and bladder neurons and double-labeled neurons were found in the reticular region and the raphe. More rostrally, bladder neurons were located in the pontine micturition center and external urethral sphincter neurons were found in the locus coeruleus and subcoeruleus. A very small number of double-labeled neurons were found in the pontine micturition center and the locus coeruleus or subcoeruleus.

Introduction

The normal process of micturition involves the accumulation and storage of urine in the urinary bladder followed by its periodic elimination via the urethra. This switch-like process requires a timely coordination between the bladder and the sphincters (internal and external) such that the sphincters close during urine storage and open during urine discharge. This process is accomplished through reflexes organized at the lumbosacral spinal cord and the brainstem [39]. Afferent impulses from the periphery pass to the spinal cord and brainstem and activate efferents to the bladder, the sphincters and the urethra. These signals travel through three nerves: the pelvic nerve the hypogastric nerve and the pudendal nerve [39]. There are somato-vesical, vesico-somatic and vesico-vesical reflexes [6], [10], [20], [22], [23], [33], [39]. These reflexes assure an efficient storage and emptying of the bladder. In an early publication Barrington [2] described five reflexes as part of the micturition process. In a much later paper expanding Barrington’s ideas [20] 12 reflexes involved in micturition were divided into four groups: storage, initiation, continuation and cessation. During urine accumulation, bladder afferents to the lumbosacral spinal cord relax the bladder via sympathetic efferents in the hypogastric nerves to beta-adrenergic receptors in the bladder body and activate the muscles of the bladder base, internal sphincter and urethra via alpha-adrenergic receptors, facilitating compliant accumulation of urine and preventing leakage. In addition to these sympathetic influences, guarding reflexes that require an intact neuraxis [30] send signals through the pudendal nerve to stimulate the external urethral sphincter to maintain continence. When the bladder has accumulated a sufficient amount of fluid, afferent outflow increases significantly and activates a spino-bulbo-spinal reflex, which initiates micturition [9], [34]. Parasympathetic outflow via the pelvic nerve contracts the bladder and signals from the brainstem inhibit the guarding reflexes [30], allowing urine to be expelled. The final cessation of micturition may also be accomplished via a reflex pathway, which includes urethral and bladder afferents [8] signaling the cessation of the passage of urine in the urethra and the collapse of the bladder. For a review of several proposed schemes describing the circuitry for storage and voiding see Kinder et al. [18].

This paper concerns the relationship between the two structures that are intimately involved in the final micturition process: the bladder and the external urethral sphincter. In an effort to examine the relationships among the relevant central nervous system neurons, we describe those that are retrogradely labeled by two immunohistochemically distinguishable pseudorabies viruses [16], [17], [19]; one injected into the bladder and the other injected into the external urethral sphincter. Pseudorabies viruses injected into these peripheral organs travel retrogradely along the nerves to infect the neurons associated with the nerves [25], [26], [27], [35]. The virus replicates within the infected neuron and is then exported, only through synapses, to neurons that innervate the infected neuron. This infection/replication/exportation process continues and, as time passes, neurons located more and more rostrally, are infected [4], [11]. Neurons labeled by either of these viruses or by both were found in the lumbosacral spinal cord and in the brainstem. A preliminary report of this work has been presented [28].